Purified total RNA from any organism. Please see our Multi-Organism Transcriptomics Application Note (data from the previous version of the kit with instruction manual v1.3.0.) or find more product information here.

This kit is not designed for metatranscriptomics, and thus the performance cannot be guaranteed for such application. In our preliminary test using total RNA extracted from our ZymoBIOMICS Microbial Community Standard (D6300), using 100 ng as input and depleting for 2 hours yielded libraries where remaining rRNA reads were around 25%. Therefore, for rRNA depletion in metatranscriptomic samples with low complexity (<10 microbial species/strains), this kit could be suitable, and we recommend using a higher amount of total RNA as input whenever possible and optionally performing the Depletion Reaction for at least 2 hours. For further information, please contact tech@zymoresearch.com.

It is possible to prepare libraries using this kit with degraded RNA with low RIN scores, such as RNA from FFPE samples, although the performance may be negatively impacted. For optimal results using degraded samples, please refer to Appendix E in the kit protocol for detailed suggestions, or contact tech@zymoresearch.com.

DNA contamination will adversely impact the accuracy and sensitivity of quantitative measures of gene expression and differential gene expression analysis. Therefore, we recommend removing DNA contamination from the RNA input prior to performing the Zymo-Seq RiboFree workflow. For extracted RNA, we recommend using the RNA Clean & Concentrator-5 (R1013), which includes DNase I treatment and subsequent clean-up. This method has been validated for use with the Zymo-Seq RiboFree workflow.

The Zymo-Seq RiboFree kit uses the input RNA as a template to drive the depletion of the reverse transcribed cDNA from the highly abundant sequences. This allows the depletion to be probe-free and universally compatible with RNA from any organism. Learn more about the probe-free depletion from this feature in Nature.

Generally, the random hexamer priming during reverse transcription and the reaction conditions during the depletion allows the production of appropriately sized inserts and final libraries for Illumina® sequencers.

Humidity and temperature vary between labs. Therefore, while we don’t recommend a specific amount of time to wait, we do recommend adding the elution buffer to the bead pellet as soon as the wet gloss dries and leaves the pellet matte in appearance. By holding the tube up to a bright light, this change is more easily seen in real time.

Avoid adding the elution buffer when the bead pellet is still wet-looking, or after the pellet cracks as both may lead to a decrease in library yield. An example image showing different levels of “dryness” is here for your reference.

Besides being included in the 96-prep RiboFree kit (R3003), the UDI indexes 1-96 are also sold separately as the Zymo-Seq UDI Primer Plate (Cat. No. D3096) for you to order.

Each UDI primer set in the Zymo-Seq UDI Primer Set (Cat. No. D3008) can be used more than once as long as the libraries with the same UDI set ARE NOT pooled and sequenced on the same sequencing lane. For more information regarding the UDI primer sets, please refer to Appendix B in the kit protocol or contact tech@zymoresearch.com.

This kit is compatible with any Illumina® sequencer. It is not directly compatible with Ion Torrent®, Oxford Nanopore®, or PacBio® platforms. 

Yes, the Zymo-Seq RiboFree depletion module is available as a stand-alone product, the Zymo-Seq RiboFree Universal cDNA Kit (R3001), and is compatible with library preparation kits that accept single-stranded DNA as an input.

There are many ways for you to be certain about which instruction manual to follow. One way is to check the cold reagent names: they are different between the two versions, and the updated version includes a reagent called "Depletion Reagent 4". Overall, compared to v1.3.0, the updated version has a lower minimum input (10 ng total RNA), a shorter depletion reaction time (1 hour for 100 ng of input), and one fewer round of bead cleanup. To learn more of the kit features since the update, or if you need an electronic copy of the previous instruction manual (v1.3.0), please contact tech@zymoresearch.com.

It is possible to prepare libraries with input lower than 10 ng, though the performance may be negatively impacted. For modifications of the protocol when using 1-9 ng of RNA as input, please refer to Appendix D in the kit protocol for detailed suggestions or contact tech@zymoresearch.com. We do NOT recommend extending the depletion reaction to run overnight.

Library yield can vary depending on factors such as the quality and quantity of the input RNA, and the cycle number of the PCR reaction for library amplification. In general, using 10 ng of Universal Human Reference RNA (RIN > 8.0) as input, the library yield is > 10nmol/L when amplified with 11 PCR cycles and eluted to 20 µL of DNA Elution Buffer.